Integrated Multi-omics Analysis of the Salivary Microbiome and Metabolome in Gestational Diabetes Mellitus

Introduction: Gestational diabetes mellitus (GDM) is associated with maternal metabolic dysregulation and may alter the oral microenvironment. Saliva provides a non-invasive matrix for monitoring host metabolic status and microbial changes; however, integrated salivary metabolomic and microbiome analyses in GDM remain limited. Methods: Saliva samples from normoglycemic pregnant women and women with GDM were analyzed using untargeted Liquid Chromatography-Mass Spectrometry（LC-MS）/MS-based metabolomics and 16S rRNA gene amplicon sequencing. Differential metabolites were identified and subjected to pathway enrichment analysis. Microbial diversity, taxonomic composition, differential taxa, and predicted functional profiles were evaluated using ASV-based bioinformatic analyses. Animals were euthanized by intraperitoneal injection(i.p.) of sodium pentobarbital (100 mg/kg). The carcasses were then subjected to centralized disposal. Results: Untargeted metabolomics identified 2,437 salivary metabolic features, among which 579 metabolites differed between the GDM and control groups, including 550 upregulated and 29 downregulated metabolites in the GDM group. These altered metabolites were mainly related to lipid and lipid-like molecules, particularly glycerophospholipids, lysophosphatidylcholines, and fatty acid derivatives. Pathway enrichment analysis suggested changes in glycerophospholipid catabolism, energy metabolism, and immune-associated processes. Microbiome analysis identified 61 shared ASVs across samples, with Bacteroidota and Firmicutes as the dominant phyla. Alpha diversity did not show clear group-level separation, whereas Bray-Curtis beta diversity indicated significant compositional differences between groups. Differential abundance and Random Forest analyses identified several candidate discriminatory taxa, including Rikenellaceae_RC9_gut_group, Helicobacter, Clostridia_UCG-014, and related bacterial lineages. Conclusion: GDM was associated with salivary lipid remodeling, immune- and energy-related metabolic alterations, and shifts in microbial composition. These findings support saliva as a promising non-invasive biofluid for exploring GDM-associated host–microbe metabolic interactions and provide candidate salivary features for future multi-omics biomarker validation.